Insulin is a hormone that plays an important role in regulating glucose metabolism. Insulin produced from Langerhans' islet β cells in the pancreas is secreted by exocytosis. The secreted insulin acts on cells having insulin receptors to stimulate glucose uptake into cells. Blood sugar levels in the body are maintained in the optimal range by the action of insulin.
Diabetes mellitus, which is a representative disease associated with insulin, is classified into Type I diabetes mellitus and Type II diabetes mellitus.
In Type I diabetes mellitus, the response to insulin is maintained and blood sugar levels can be controlled by the administration of insulin formulations (e.g., animal insulin preparations extracted from the bovine or swine pancreas; human insulin preparations synthesized by a genetic engineering technique using Escherichia coli or a yeast), or by the administration of insulin secretagogues (for example, sulfonylureas (e.g., tolbutamide, glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide, glyclopyramide, glimepiride, glipizide, glybuzole, etc.), repaglinide, senaglinide, nateglinide, mitiglinide, etc.).
The insulin preparations are classified as rapid-acting, regular-acting, intermediate-acting, long-acting, mixed (rapid-acting+intermediate-acting, regular-acting+intermediate-acting), long-acting soluble preparations, and the like, based on time of action. These insulin preparations are appropriately administered depending on the symptoms and conditions of the patient.
As described above, a variety of drugs are known as insulin secretagogues but these drugs involve problems that preprandial hypoglycemia, postprandial hyperglycemia, etc. tend to occur.
Extracellular release (secretion) of a secretory protein is mediated by a mechanism that trafficking vesicles fuse with cell membranes (exocytosis). The total internal reflection fluorescence method (TIRF method) and the fluorescence imaging method (two-photon molecular excitation method) using the excitation of two photons are known as techniques for the visualization of exocytosis.
It has been reported that secretion of the fused protein of enhanced GFP, which is one of fluorescent proteins, with insulin (insulin-EGFP) was observed by the total internal reflection fluorescence method (J. Biol. Chem. 277, 3805-3808 (2002)). More specifically, the phenomenon of exocytosis with the trafficking vesicles containing insulin-EGFP fusion protein was observed from the adhesive part of cells on the side of a glass-bottom dish.
It has been reported that fluorescent dyes were permeated into the spaces where cells are closely adhered to each other, and the secretory vesicles containing insulin fused the cell membranes to form the omega-type structure was observed by the two-photon molecular excitation method (Science, 297, 1349-1352 (2002)).
However, both the total internal reflection fluorescence method and the two-photon molecular excitation method have problems that localization of the exocytotic sites on the whole cells cannot be identified and the secreted proteins cannot be quantified, etc.
It has been reported that the exocytotic secretion of luciferase was observed by the bioluminescence imaging method, using Vargula luciferase (Proc. Natl. Acad. Sci., USA, 89, 9584-9587 (1992)).
It has been reported that the process for the secretion of the fused protein (DBHsp-GLase) of the signal peptide sequence (DBHsp) of human DBH (dopamine β-hydroxylase) with Gaussia luciferase (GLase) was observed by the bioluminescence imaging method, (FEBS Letters, 581, 4551-4556 (2007)).
However, no report is known of observation of the exocytotic secretion of functional proteins and functional polypeptides such as hormones, growth factors, etc., by the bioluminescence imaging method.
Matrix metalloproteinase (MMP) is a superfamily of zinc-dependent endopeptidases and involved in the degradation of the major components of extracellular matrix and connective tissues that inhibit motility of cells.
MMP, especially a gelatinase is known to be associated with metastasis and diffusion of cancers. For example, MMP-2 and MMP-9, which are gelatinases, are known to rise in a particular tumor promoting event. MMP-2 and MMP-9 degrade type IV collagen as the main components of the basal membrane and denatured collagen (gelatin) to induce tumor metastasis. It is also known that disruption of vascular membranes mainly composed of type IV collagen plays an important role in tumor metastasis.